Biomolecules of medical or industrial importance, which may be proteins, glycoproteins, lipoproteins, polysaccharides, lipids or nucleic acids, are produced by a wide variety of methods including chemical synthesis; secretion into culture medium by naturally occurring or recombinantly transformed bacteria, yeasts, fungi, insect cells, and mammalian cells; accumulation in cultured cells (e.g., in inclusion bodies); secretion from genetically engineered organisms (e.g., in the milk of transgenic mammals); and recovery from biological sources such as urine, blood, milk, plant infusions, fungal extracts, and the like. Most biomolecules thus produced are of little use, however, without purification away from other elements (i.e., "impurities") present in the solutions in which they are produced or without concentration of the biomolecule so that it comprises a much larger fraction of the solution.
Chromatography is a dominant purification and concentration technique used in the large-scale isolation of biomolecular targets. With a properly designed series of chromatographic steps, a single biomolecular component can be isolated from a complex mixture with contaminants that range from inorganic salts to incorrectly folded or partially degraded forms of the target macromolecule itself. At the same time, chromatographic purification is a major (and often the largest single) cost in the manufacture of a biomolecule product. Typical biotlerapeutic purifications require from two to six or more chromatographic steps, utilizing size-exclusion, ion-exchange, hydrophobic interaction, and affinity chromatographic modes.
While size-exclusion is often used for buffer exchange or as a final step to remove aggregated material, and ion-exchange and hydrophobic interaction are used to concentrate the product and remove major impurities, none of these chromatographies can approach the dramatic singlestep increases in purity achieved using affinity chromatography. Narayanan (1994), for instance, reported a 3000-fold increase in purity through a single affinity chromatography step.
Affinity chromatography is not, however, a commonly used technique in largescale production of biomolecules. The ideal affinity chromatography ligand must, at acceptable cost, (1) capture the target biomolecule with high affinity, high capacity, high specificity, and high selectivity; (2) either not capture or allow differential elution of other species (impurities); (3) allow controlled release of the target under conditions that preserve (i.e., do not degrade or denature) the target; (4) permit sanitization and reuse of the chromatography matrix; and (5) permit elimination or inactivation of any pathogens. However, finding high-affinity ligands of acceptable cost that can tolerate the cleaning and sanitization protocols required in pharmaceutical manufacturing has proved difficult (see, Knight, 1990).
Although far from ideal, dyes (such as cibachron blue) and proteins of known affinity (such as Protein A) have been employed extensively in affinity chromatography. These materials, however, cannot be adapted to new targets and therefore lack the flexibility to be more widely used.
Murine monoclonal antibodies (MAbs) also have been used as affinity ligands. MAbs can be readily generated, and new MAb ligands specific for a new target molecule can be obtained, giving MAb technology a degree of flexibility to meet the individual requirements of a particular manufacturer. Monoclonal antibodies, on the other hand, are not without drawbacks in the field of affinity chromatography: MAbs are expensive to produce, and they are prone to leaching and degradation under the cleaning and sanitization procedures associated with purification of biomolecules, leading MAb-based affinity matrices to lose activity quickly (see, Narayanan, 1994; Boschetti, 1994). In addition, although MAbs can be highly specific for a target, the specificity is often not sufficient to avoid capture of impurities that are closely related to the target. Moreover, the binding characteristics of MAbs are determined by the immunoglobulin repertoire of the immunized animal, and therefore practitioners must settle for the binding characteristics they are dealt by the animal's immune system, i.e., there is little opportunity to optimize or select for particular binding or elution characteristics using only MAb technology. Finally, the molecular mass per binding site (25 kDa to 75 kDa) of MAbs and even MAb fragments is quite high.
Thus, there is a continuing need to develop less expensive, more serviceable and more tailored affinity ligands for particular biomolecular targets. Specifically, there is a need for affinity ligands that more closely approach the characteristics of the ideal affinity ligand described above, that not only bind to a given target molecule with high affinity but also release the target under desirable or selected conditions, that are able to discriminate between the target and other components of the solution in which the target is presented, and/or that are able to endure cleaning and sanitization procedures to provide regenerable, reusable chromatographic matrices.
Such affinity ligands and methods for obtaining them are provided herein.